The invention relates generally to the identification of genes responsible for the virulence of Staphylococcus bacteria, thereby allowing the identification of new anti-bacterial agents that target these virulence genes and their products and the provision of novel S. aureus mutants useful in vaccines.
The staphylococci, of which Staphylococcus aureus is the most important human pathogen, are hardy, gram-positive bacteria that colonize the skin of most humans. Staphylococcal strains that produce coagulase are designated S. aureus; other clinically important coagulase-negative staphylococci are S. epidermidis and S. saprophyticus. When the skin or mucous membrane barriers are disrupted, staphylococci can cause localized and superficial infections that are commonly harmless and self-limiting. However, when staphylococci invade the lymphatics and the blood, potentially serious complications may result, such as bacteremia, septic shock, and serious metastatic infections, including endocarditis, arthritis, osteomyelitis, pneumonia and abscesses in virtually any organ. Certain strains of S. aureus produce toxins that cause skin rashes, food poisoning, or multisystem dysfunction (as in toxic shock syndrome). S. aureus and S. epidermidis together have become the most common cause of nosocomial non-urinary tract infection in U.S. hospitals. They are the most frequently isolated pathogens in both primary and secondary bacteremias and in cutaneous and surgical wound infections. See generally Harrison""s Principles of Internal Medicine, 13th ed., Isselbacher et al., eds., McGraw-Hill, New York (1994), particularly pages 611-617.
Transient colonization of the nose by S. aureus is seen in 70 to 90 percent of people, of which 20 to 30 percent carry the bacteria for relatively prolonged periods of time. Independent colonization of the perineal area occurs in 5 to 20 percent of people. Higher carriage rates of S. aureus have been documented in persons with atopic dermatitis, hospital employees, hospitalized patients, patients whose care requires frequent puncture of the skin, and intravenous drug abusers.
Infection by staphylococci usually results from a combination of bacterial virulence factors and a diminution in host defenses. Important microbial factors include the ability of the staphylococcus to survive under harsh conditions, its cell wall constituents, the production of enzymes and toxins that promote tissue invasion, its capacity to persist intracellularly in certain phagocytes, and its potential to acquire resistance to antimicrobials. Important host factors include an intact mucocutaneous barrier, an adequate number of functional neutrophils, and removal of foreign bodies or dead tissue.
Cell wall components of S. aureus include a large peptidoglycan complex that confers rigidity on the organism and enables it to survive under unfavorable osmotic conditions, a unique teichoic acid linked to peptidoglycan, and protein A, which is found both attached to peptidoglycan over the outermost parts of the cell and released in soluble form. Proteins designated femA and femB are involved in the formation of cell wall peptidoglycan pentaglycine cross-bridges and are factors in methicillin resistance. [Berger-Bachi et al., Mol. Gen. Genet., 219:263-269 (1989).] S. aureus also has specific receptors for laminin and fibronectin that may mediate the organism""s spread through the bloodstream to other tissues. Both peptidoglycan and teichoic acid are capable of activating the complement cascade via the alternative pathway. S. aureus also appears to activate tissue factor in the coagulation pathway.
Certain enzymes produced by S. aureus may play a role in virulence. Catalase degrades hydrogen peroxide and may protect the organism during phagocytosis. Coagulase is present in both soluble and cell-bound forms and causes plasma to clot by formation of thrombin-like material. The high correlation between coagulase production and virulence suggests that this substance is important in the pathogenesis of staphylococcal infections, but its precise role as a determinant of pathogenicity has not been determined. Many strains also produce hyaluronidase, an enzyme that degrades hyaluronic acid in the connective tissue matrix and that may promote spreading of infection. A trypsin-like protease from some strains enhances influenza virus infection by proteolytic cleavage of the viral precursor hemagglutinin into its active fragments and may contribute to the morbidity of such coinfections.
S. aureus produces numerous extracellular exotoxins that have been implicated in disease processes. The exfoliatin toxins A and B, the staphylococcal enterotoxins, and the toxic shock syndrome toxin, TSST-1, belong to the growing family of microbial superantigens that activate T cells and monocytes/macrophages, resulting in the production of cytokines that mediate local or systemic effects depending on the amount of toxin formed, the immune status of the host, and the access of the toxin to the circulation. The exfoliatin toxins mediate the dermatologic manifestations of the staphylococcal scalded-skin syndrome and bullous impetigo. These toxins cause intraepidermal cleavage of the skin at the stratum granulosum, leading to bullae formation and denudation. Seven distinct enterotoxins (A, B, C1, C2, C3, D, and E) have been implicated in food poisoning due to S. aureus. These toxins enhance intestinal peristalsis and appear to induce vomiting by a direct effect on the central nervous system. Toxic shock syndrome (TSS) is most conunonly mediated by TSST-1, which is present in 5 to 25 percent of clinical isolates of S. aureus. TSS is also mediated less frequently by enterotoxin B and, rarely, enterotoxin C1.
S. aureus produces other toxins whose role in virulence is incompletely understood. Four different red blood cell hemolysins, which are designated alpha, beta, gamma, and delta toxins, have been identified. Alpha toxin also causes necrosis of the skin when injected subcutaneously into animals, while delta toxin also inhibits water absorption in the intestines and may play a role in the acute watery diarrhea seen in some cases of staphylococcal infection. Leukocidin lyses granulocyte and macrophage membranes by producing membrane pores permeable to cations.
The agr, xpr, sae and sar genes have been identified as being involved in the regulation of staphylococcal exotoxins. See U.S. Pat. No. 5,587,228 and International Patent Publication Nos. WO 96/10579 and WO 97/11690. Of interest is the report in WO 97/11690 of screening for inhibitors of these regulatory systems.
Staphylococci can invade the skin or mucosa through plugged hair follicles and sebaceous glands or areas traumatized by burns, wounds, abrasions, insect bites, or dermatitis. Staphylococci often colonize prosthetic devices and intravenous catheters; S. aureus infection of the vascular access site is a major cause of morbidity and death among patients on hemodialysis. Colonization and invasion of the lungs may occur with endotracheal intubation, or when the lungs"" clearance mechanisms are depressed, e.g., after viral infections, after aspiration, or in patients with cystic fibrosis. Mucosal damage to the gastrointestinal tract following cytotoxic chemotherapy or radiotherapy predisposes to invasion from that site.
Once the skin or mucosa have been breached, local bacterial multiplication is accompanied by inflammation, neutrophil accumulation, tissue necrosis, thrombosis and fibrin deposition at the site of infection. Later, fibroblasts create a relatively avascular wall about the area. When host mechanisms fail to contain the cutaneous or submucosal infection, staphylococci may enter the lymphatics and the bloodstream. Common sites of metastatic spread include the lungs, kidneys, cardiac valves, myocardium, liver, spleen, bones and brain.
Bacteremia due to S. aureus may arise from any local infection, at either extravascular (cutaneous infections, burns, cellulitis, osteomyelitis, arthritis) or intravascular foci (intravenous catheters, dialysis access sites, intravenous drug abuse). Rarely, patients with bacteremia die within 12 to 24 hours with high fever, tachycardia, cyanosis, and vascular collapse. Disseminated intravascular coagulation may produce a disease mimicking meningococcemia. Commonly, the disease progresses more slowly, with hectic fever and metastatic abscess formation.
A major complication of S. aureus bacteremia is endocarditis. S. aureus is the second most common cause of endocarditis and the most common cause among drug addicts. The disease is typically acute, with high fever, progressive anemia, and frequent embolic and extracardiac septic complications. Valve ring and myocardial abscesses are common. The mortality rate is 20 to 30 percent.
Staphylococcal scalded-skin syndrome (SSSS) is a generalized exfoliative dermatitis that is a complication of infection by exfoliatin toxin-producing stranis of S. aureus. The disease typically occurs in newborns (Ritter""s disease) and in children under the age of five. A scarlatiniform rash begins in the perioral area, becomes generalized over the trunk and extremities, and finally desquamates. The disease may consist of rash alone (staphylococcal scarlet fever), or large, flaccid bullae develop that may be localized (more common in adults) or generalized. The bullae burst, resulting in red, denuded skin resembling a burn. Most adults with SSSS are immunosuppressed or have renal insufficiency. Blood cultures are frequently positive, and mortality is significant.
Toxic shock syndrome (TSS) is a multisystem disease mediated by toxins (generally TSST-1, and less frequently enterotoxins B and C1) produced by certain strains of S. aureus. It was first described in children, but in 1980 became epidemic among young women, with onset during menstruation. The diagnosis of TSS is based on clinical criteria that include high fever, a diffuse rash that desquamates on the palms and soles over the subsequent one or two weeks, hypotension that may be orthostatic, and evidence of involvement in three or more organ systems. Such involvement commonly includes gastrointestinal dysfunction (vomiting or diarrhea), renal or hepatic insufficiency, mucous membrane hyperemia, thrombocytopenia, myalgias with elevated creatine phosphokinase (CK) levels, and disorientation with a normal cerebrospinal fluid examination. The mortality rate of TSS is three percent.
S. aureus causes approximately three percent of community-acquired bacterial pneumonias. This disease occurs sporadically except during influenza outbreaks, when staphylococcal pneumonia is relatively more common, although still less frequent than pneumococcal pneumonia. Primary staphylococcal pneumonia in infants and children frequently presents with high fever and cough. Multiple thin-walled abscesses are seen on the chest X-ray, and empyema formation is common. In older children and healthy adults, staphylococcal pneumonia is generally preceded by an influenza-like respiratory infection. Onset of staphylococcal involvement is abrupt, with chills, high fever, progressive dyspnea, cyanosis, cough, pleural pain, and sometimes bloody sputum. Staphylococcal pneumonia is seen more frequently in patients with cystic fibrosis, in intubated patients in intensive care units and in debilitated patients who are prone to aspiration.
S. aureus is responsible for the majority of cases of acute osteomyelitis. Although the disease is most common in people under the age of 20, it is becoming increasingly prevalent in adults over 50, particularly with involvement of the spine. A primary portal of entry is frequently not identified, although many patients give a history of preceding trauma to the involved area. Once established, infection spreads through the bone to the periosteum or along the marrow cavity. Rarely, the joint capsule is penetrated, producing pyogenic arthritis. Osteomyelitis in children may present as an acute process beginning abruptly with chills, high fever, nausea, vomiting, and progressive pain at the site of bony involvement.
S. aureus causes 1 to 9 percent of cases of bacterial meningitis and 10 to 15 percent of brain abscesses. Most commonly, the bacteria are spread from a focus outside the central nervous system, typically from infective endocarditis, by extension from a paraspinal or parameningeal abscess, or by nosocomial infection following neurosurgical procedures. Over 50 percent of epidural abscesses are due to S. aureus; up to half of these cases may be associated with vertebral osteomyelitis. Patients present with either acute or chronic back pain, usually with low-grade fever and malaise. The onset of radicular pain is an ominous sign that the disease may progress to neurologic dysfunction and ultimate paralysis.
Antimicrobial resistance by staphylococci favors their persistence in the hospital environment. Over 90 percent of both hospital and community strains of S. aureus causing infection are resistant to penicillin. This resistance is due to the production of xcex2-lactamases enzymes; the genes for these enzymes are usually carried by plasmids. Infections due to organisms with such acquired resistance can sometimes be treated with penicillinase-resistant xcex2-lactam antimicrobial agents. However, the true penicillinase-resistant S. aureus organisms, called methicillin-resistant S. aureus (MRSA), are resistant to all the xcex2-lactam antimicrobials as well as the cephalosporins. MRSA resistance is chromosomally mediated and involves production of an altered penicillin-binding protein (PBP 2a or PBP 2xe2x80x2) with a low binding affinity for xcex2-lactams. MRSA frequently also have acquired plasmids mediating resistance to erythromycin, tetracycline, chloramphenicol, clindamycin, and aminoglycosides. MRSA have become increasingly common worldwide, particularly in tertiary-care referral hospitals. In the United States, approximately 5 percent of hospital isolates of S. aureus are methicillin-resistant.
Thus, there continues to exist a need for new agents useful for treating bacterial infections, particularly those caused by antibioic-resistant bacteria, and for methods of identifying such new agents. Such methods ideally would identify agents that are unrelated to existing antimicrobials and that target different aspects of staphylococcal invasion of and replication in the host, compared to existing antimicrobials.
The present invention relates generally to the identification of genes responsible for the virulence of Staphylococcus bacteria, thereby allowing the identification of new anti-bacterial agents that target these virulence genes and their products and the provision of novel S. aureus mutants useful in vaccines.
According to one aspect of the present invention, methods are provided for identifying anti-bacterial agents that target the function of staphylococcal virulence genes or gene products. Such methods include assaying potential agents for the ability to interfere with expression of virulence gene products represented by the DNA sequences set forth in any one of SEQ ID NOS: 1 through 94, or assaying potential agents for the ability to interfere with the function of a bacterial protein encoded in whole or in part by a DNA sequence set forth in any one of SEQ ID NOS: 1 through 94 or the complementary strand thereof, followed by identifying agents that are positive in such assays.
The use of a number of different assays is contemplated according to this aspect of the invention. When the function of the virulence gene product is known or predicted by sequence similarity to a known gene product, potential inhibitors can be screened in enzymatic or other types of assays keyed to the function of the gene product. When the virulence gene product is known or predicted by sequence similarity to a known gene product to interact with another protein or nucleic acid, inhibitors of this interaction can be screened directly in binding assays or using the two-hybrid assay. Other assays may be used when a ligand for the virulence gene product is not known, including two-hybrid screening assays that identify gene products that interact with target protein, assays that identify ligands of target protein through measuring of direct binding of test ligand to target protein, and assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.
In another aspect of this invention, methods are provided for assaying potential agents for the ability to interfere with expression of or function of virulence gene products, wherein the virulence genes encoding these products are obtainable by identification through signature-tagged mutagenesis as defined herein and exemplified in Example 1.
According to a further aspect of this invention, novel anti-bacterial agents identified by the methods described herein are provided, as well as methods for treating a subject suffering from infection with staphylococci involving administration of such novel anti-bacterial agents. In particular, agents that interfere with the expression of virulence gene products include anti-sense polynucleotides that are complementary to the virulence gene sequences. Agents that interfere with the function of virulence gene products include variants of virulence gene products, ligands of these virulence gene products and variants thereof, and enzyme inhibitors (where the product is an enzyme).
Yet a further aspect of this invention provides Staphylococcus aureus organisms containing a functional mutation in a gene represented by any one of SEQ ID NOS: 1 through 94, said functional mutation resulting in a reduction in virulence of the organism. Also contemplated are vaccine compositions comprising such mutated S. aureus organisms, optionally comprising a suitable adjuvant and a pharmaceutically acceptable diluent or carrier.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently prepared embodiments thereof.
xe2x80x9cVirulence genes,xe2x80x9d as used herein, are genes whose function or products are required for successful establishment and/or maintenance of bacterial infection in a host animal. Thus, virulence genes and/or the proteins encoded thereby are involved in pathogenesis in the host organism, but may not be necessary for growth in vitro. Since antibiotics are typically screened in vitro, identification of these in vivo virulence genes provides a means for discovering new antimicrobials with different targets and mechanisms of action compared to existing antibiotics. There may be 50 to 100 virulence genes in S. aureus [see Groisman and Ochman, Trends Microbiol. Sci., 2:289-294 (1984) (discussing Salmonella virulence genes); Muhldorfer and Hacker, Microb. Pathogenesis, 16:171-181 (1994) (discussing E. coli virulence genes).
xe2x80x9cSignature-tagged mutagenesis,xe2x80x9d as used herein, is a method generally described in International Patent Publication No. WO 96/17951, incorporated herein by reference, and includes, for example, a method for identifying S. aureus genes required for virulence in a murine model of bacteremia. In this method, each insertional mutation carries a different DNA signature tag which allows mutants to be differentiated from each other. The tags comprise 40-bp variable central regions flanked by invariant xe2x80x9carmsxe2x80x9d of 20-bp which allow the central portions to be co-amplified by polymerase chain reaction (PCR). Tagged mutant strains are assembled in microtitre dishes, then combined to form the xe2x80x9cinoculum poolxe2x80x9d for infection studies. At an appropriate time after inoculation, bacteria are isolated from the animal and pooled to form the xe2x80x9crecovered pool.xe2x80x9d The tags in the recovered pool and the tags in the inoculum pool are separately amplified, labelled, and then used to probe filters arrayed with the different tags representing the mutants in the inoculum. Mutants with attenuated virulence are those with tags that give hybridization signals when probed with tags from the inoculum pool but not when probed with tags from the recovered pool.
Signature-tagged mutagenesis allows a large number of insertional mutant strains to be screened simultaneously in a single animal for loss of virulence. Screening thirteen pools of 96 mutant S. aureus strains resulted in the identification of 50 strains with reduced virulence, many of which were confirmed to be attenuated in virulence by subsequent analysis of individual mutants. The nucleotide sequences of the regions flanking the transposon insertion points of these mutants were analyzed by searching DNA and protein sequence databases to identify the genes inactivated by the insertion of the transposon.
On the basis of these searches many of the virulence genes may be grouped into different classes. The first class encodes proteins involved in cell surface metabolism (e.g., P2C73, P11C29, P13C83, P9B65, P10B89). Both femA and femB, which are involved in the formation of cell wall peptidoglycan pentaglycine cross bridges, were identified as virulence genes. Mutant P2C73 contains a transposon insertion in a previously unknown gene that shares significant similarity to femB. Mutant P14C15 contains a transposon insertion in a gene whose product is similar to aspartate semialdehyde dehydrogenase (Asd) from different bacteria, with the highest level of similarity to Asd from Bacillus subtilis. Asd is a key enzyme in the biosynthesis of methionine, threonine, isoleucine, lysine and diaminopimelic acid (DAP), which is an important component of cell wall peptidoglycan.
The second class encodes enzymes involved in cellular biosynthetic pathways (e.g., P9B74, P5C4, P9B66, P14C15, P13B26, P7C18, P15C31, P10B18, P6B18, P10B66, P10C34, P12C3). Deduced protein products of two genes (mutants P7C18 and P13B26) show strong similarity to B. subtilis LysA and ThrB. These enzyzmles, like Asd, are involved in aspartate metabolism. LysA is diaminopimelate decarboxylase, which converts diaminopimelate to lysine, and ThrB phosphorylates homoserine before conversion into threonine. Transposon insertions were also obtained in genes homologous to Methanococcus jannaschii trpA, Lactococcus lactis trpB and L. lactis trpD. These genes encode enzymes of the tryptophan biosynthetic pathway: the alpha chain of tryptophan synthetase, the beta chain of tryptophan synthetase, and anthranilate phosphoribosyltransferase, respectively. The gene mutated in P15C31 is a homolog of L. lactis purL encoding phosphoribosylformylglycinamidine decarboxylase, an enzyme of the purine biosynthetic pathway. Mutant P9B66 contains an insertion in a gene whose product is similar to peptide methionine sulphoxide reductases.
A third class of genes are those encoding components of the TCA cycle (e.g., mutants P4C27, P4C52, P10B2, P10C20, P12C32). Strains P10B2 and P12C32 carry mutations in genes for a subunit of the oxoglutarate dehydrogenase complex and aconitase, respectively.
The fourth class is composed of genes whose products are similar to a oligopeptide transport proteins of the ATP-binding cassette (ABC) transporter superfamily (e.g., mutants P7C26, P10C15, P5C3, P11C66, P5C34). Oligopeptide transport is important for peptide utilization and the proteolytic system in lactococci. In Group A streptococci, Opp proteins are involved not only in obtaining nutrients but also in adherence, protease production and processing of secreted proteins.
The fifth class of genes are involved in cellular regulatory and repair processes (e.g., mutants P4C15, P13B74, P13C72, P10B30, P6C63, P14B25). Mutant P4C15 and P6C63 contain insertions in S. aureus MarR/LuxR-like regulatory proteins. MarR and LuxR belong to a family of transcription regulators and these MarR/LuxR-like proteins likely have a similar function in S. aureus. In Streptococcus pneumoniae, Neisseria gonorrhoeae and Escherichia coli this enzyme helps to maintain surface adhesins in their functional oxidative state. Mutant P10B30 is associated with a transposon insertion in a gene with a product similar to the ATP-dependent Clp protease of E. coli. The Clp stress response system for intracellular protein degradation is widely conserved in bacteria and components of the system are important for virulence of Listeria monocytogenes and S. typhimurium. Mutants P13B74 and P13C72 have stem-loop termination sequences which possibly function in transcription termination.
The identification of these genes as virulence genes renders them useful in methods of identifying new anti-bacterial agents according to the present invention. Such methods include assaying potential agents for the ability to interfere with expression of virulence gene products represented by the DNA sequences set forth in any one of SEQ ID NOS: 1 through 94 (i.e., the genes represented by DNA sequences of SEQ ID NOS: 1 through 94 encode the virulence gene product, or the DNA sequences of SEQ ID NOS: 1 through 94 are adjacent to the gene encoding the virulence gene product, or are involved in regulation of expression of the virulence gene product), or assaying potential agents for the ability to interfere with the function of a bacterial protein encoded in whole or in part by a DNA sequence set forth in any one of SEQ ID NOS: 1 through 94 or the complementary strand thereof, followed by identifying agents that are positive in such assays. Polynucleotides and polypeptides useful in these assays include not only the genes and encoded polypeptides as disclosed herein, but also variants thereof that have substantially the same activity as the wild-type genes and polypeptides. xe2x80x9cVariants,xe2x80x9d as used herein, includes polynucieotides or polypeptides which contain one or more deletions insertions or substitutions, as long as the variant retains substantially the same activity of the wild-type polynucleotide or polypeptide. With regard to polypeptides, deletion variants are contemplated to include fragments lacking portions of the polypeptide not essential for biological activity, and insertion variants are contemplated to include fusion polypeptides in which the wild-type polypeptide or fragment thereof have been fused to another polypeptide.
The virulence genes may be cloned by PCR, using S. aureus genomic DNA as the template. For ease of inserting the gene into expression vectors, PCR primers are chosen so that the PCR-amplified gene has a restriction enzyme site at the 5xe2x80x2 end preceding the initiation codon ATG, and a restriction enzyme site at the 3xe2x80x2 end after the termination codon TAG, TGA or TAA. If desirable, the codons in the gene are changed, without changing the amino acids, according to E. coli codon preference described by Grosjean and Fiers, Gene, 18:199-209 (1982), and Konigsberg and Godson, Proc. Natl. Acad. Sci. (USA), 80:687-691 (1983). Optimization of codon usage may lead to an increase in the expression of the gene product when produced in E. coli. If the gene product is to be produced extracellularly, either in the periplasm of E. coli or other bacteria, or into the cell culture medium, the gene is cloned without its initiation codon and placed into an expression vector behind a signal sequence. For example, cloning and expression of the femA gene is described in Example 3 below.
To simplify the protein purification process, a purification tag may be added either at the 5xe2x80x2 or 3xe2x80x2 end of the gene coding sequence. Commonly used purification tags include a stretch of six histidine residues (U.S. Pat. Nos. 5,284,933 and 5,310,663), a streptavidin-affinity tag described by Schmidt and Skerra, Protein Engineering, 6:109-122 (1993), a FLAG peptide [Hopp et al., Biotechnology, 6:1205-1210 (1988)], glutathione S-transferase [Smith and Johnson, Gene, 67:31-40 (1988)], and thioredoxin [LaVallie et al., Bio/Technology, 11:187-193 (1993)]. To remove these peptide or polypeptides, a proteolytic cleavage recognition site may be inserted at the fusion junction. Commonly used proteases are factor Xa, thrombin, and enterokinase.
Proteins are produced in any number of well-known prokaryotic or eukaryotic expression systems using known promoters, vectors, and hosts. Any suitable host cell may be used for expression of the gene product, such as E. coli, other bacteria, including Bacillus and S. aureus, yeast, including Pichia pastoris and Saccharomyces cerevisiae, insect cells, or mammalian cells, including CHO cells, utilizing suitable vectors known in the art. Proteins may be produced directly or fused to a peptide or polypeptide, and either intracellularly or extracellularly by secretion into the periplasmic space of a bacterial cell or into the cell culture medium. Secretion of a protein requires a signal peptide (also known as pre-sequence); a number of signal sequences from prokaryotes and eukaryotes are known to function for the secretion of recombinant proteins. During the protein secretion process, the signal peptide is removed by signal peptidase to yield the mature protein.
The virulence gene products produced by the methods described above are used in high throughput assays to screen for inhibitory agents. The sources for potential agents to be screened are chemical compound libraries, fermentation media of Streptomycetes, other bacteria and fungi, and cell extracts of plants and other vegetations. For proteins with known enzymatic activity, assays are established based on the activity, and a large number of potential agents are screened for ability to inhibit the activity. For proteins that interact with another protein or nucleic acid, binding assays are established to measure such interaction directly, and the potential agents are screened for ability to inhibit the binding interaction.
Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields and Song, Nature, 340:245-246 (1989), and Fields and Sternglanz, Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference. The two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA-binding proteins, to identify peptides that bind to a protein, and to screen for drugs. The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA-binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast. The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a protein X, and (2) an activation domain fused to a protein Y. The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene. The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of protein X and protein Y tethers the activation domain to the UAS, activating transcription of the reporter gene. When the virulence gene product (protein X, for example) is already known to interact with another protein or nucleic acid (protein Y, for example), this assay can be used to detect agents that interfere with the interaction of X and Y. Expression of the reporter gene is monitored as different test agents are added to the system; the presence of an inhibitory agent results in lack of a reporter signal.
When the function of the virulence gene product is unknown and no ligands are known to bind the gene product, the yeast two-hybrid assay can also be used to identify proteins that bind to the gene product. In an assay to identify proteins that bind to protein X (the target protein), a large number of hybrid genes each containing a different protein Y are produced and screened in the assay. Typically, Y is encoded by a pool of plasmids in which total cDNA or genomic DNA is ligated to the activation domain. This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of protein Y. The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
Other assays may be used to search for agents that bind to the target protein. One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat. No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states. When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state. Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules. For example, use of femA in this method of screening for potential ligands is described in Example 3 below.
Another method for identifying ligands for a target protein is described in Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporated herein by reference. This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein. Agents that bind to the target protein are separated from other library components by centrifugal ultrafiltration. The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy. This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.
The inhibitors/binders identified by the initial screens are evaluated for their effect on virulence in vivo mouse models of S. aureus infections. Models of bacteremia, endocarditis, septic arthritis, soft tissue abscess or pneumonia may be utilized. Inhibitors/binders that interfere with bacterial virulence are capable of preventing the establishment of an infection or reversing the outcome of an infection once it is established.
The identification of S. aureus virulence genes also provides for microorganisms exhibiting reduced virulence, which are useful in vaccines. Such microorganisms include the S. aureus mutants generated in Example 1 below and other S. aureus mutants containing at least one functional mutation in a gene represented by any one of SEQ ID NOS: 1 through 94. The reduced virulence of these organisms and their immunogenicity may be confirmed by administration to a subject. While it is possible for an avirulent microorganism of the invention to be administered alone, one or more of such mutant microorganisms are preferably administered in a vaccine composition containing suitable adjuvant(s) and pharmaceutically acceptable diluent(s) or carrier(s). The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the avirulent microorganism of the invention and not deleterious to the subject to be immunized. Typically, the carriers will be water or saline which will be sterile and pyrogen free. The subject to be immunized is a subject needing protection from a disease caused by a virulent form of S. aureus. 
Any adjuvant known in the art may be used in the vaccine composition, including oil-based adjuvants such as Freund""s Complete Adjuvant and Freund""s Incomplete Adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), Biostim(trademark) (e.g., 01K2), the xe2x80x9cIscomsxe2x80x9d of EP 109 942, EP 180 564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic(copyright) polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). Recently, an alternative adjuvant consisting of extracts of Amnycolata, a bacterial genus in the order Actinomycetales, has been described in U.S. Pat. No. 4,877,612. Additionally, proprietary adjuvant mixtures are commercially available. The adjuvant used will depend, in part, on the recipient organism. The amount of adjuvant to administer will depend on the type and size of animal. Optimal dosages may be readily determined by routine methods.
The vaccine compositions optionally may include pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma.
The vaccine compositions can be packaged in forms convenient for delivery. The compositions can be enclosed within a capsule, sachet, cachet, gelatin, paper or other container. These delivery forms are preferred when compatible with entry of the immunogenic composition into the recipient organism and, particularly, when the immunogenic composition is being delivered in unit dose form. The dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.
The vaccine compositions may be introduced into the subject to be immunized by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, or subcutaneous injection; by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; or by surgical implantation, e.g., embedded under the splenic capsule or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time.
It will be appreciated that the vaccine of the invention may be useful in the fields of human medicine and veterinary medicine. Thus, the subject to be immunized may be a human or an animal, for example, cows, sheep, pigs, horses, dogs and cats, and poultry such as chickens, turkeys, ducks and geese.
Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Example 1 addresses identification of S. aureus genes associated with virulence by generating S. aureus mutants that contain a chromosomal insertion of a signature-tagged transposon and identifying the mutants with reduced virulence. Example 2 addresses confirmation of the attenuated virulence of individual mutants. Example 3 addresses use of the virulence genes and gene products in assays for screening potential agents for anti-bacterial activity.